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Extra info for Current Protocols in Protein Science
Continue stirring for a further 20 min, then centrifuge 20 min at 14,000 × g, 4°C. 4. 5 Current Protocols in Protein Science Supplement 29 Perform chromatography on GTP-Sepharose 13. 5–cm chromatography column packed to 22 cm with Sephadex G-25, equilibrated with affinity chromatography buffer. The next chromatographic steps should be performed as quickly as possible, since the enzyme is slightly unstable in the affinity chromatography buffer. 14. Apply the enzyme solution to a 6 × 2–cm chromatography column packed to 4 cm with hydrazide-Sepharose 4B equilibrated with affinity chromatography buffer.
C. Also prepare sample containing no bis-NAD+ (to which water is added in place of the coenzyme derivative) as well as a control tube that contains no bis-NAD+ or glutarate, so that the percentage inhibition resulting from the presence of the glutarate can be calculated. 4. A 1 M solution of bis-NAD+ will give an absorbance, in a 1-cm light-path cuvette, of 42,800, and each mole of bis-NAD+ contains 2 NAD+ equivalents. 25 Current Protocols in Protein Science Supplement 29 4. Allow the tubes to stand at 4°C for at least 30 min (or overnight, if required), then microcentrifuge 5 min at maximum speed (∼13,000 × g), 4°C.
Concentrate the product-containing fractions to ∼3 to 5 ml by rotary evaporation at 35°C and store at –20°C. For longer-term storage (>7 to 10 days) the material may be lyophilized and stored in a vacuum desiccator over silica gel at –20°C. 5 Outline of the steps involved in the synthesis of bis-NAD+. Full details of the steps involved are given in Support Protocol 7. , Wilson and Walker, 2000; Hahn-Deinstrop, 2000). 8/1-propanol. System B: 5:3 (v/v) isobutyric acid/1 M aqueous NH3, saturated with disodium EDTA.